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Single-Base tRNA Modification Sequencing
Single-Nucleotide tRNA m5C Modification Seq(BS-seq)
BS-seq is an antibody-free, base-resolution approach that maps 5-methylcytidine (m5C) on tRNA. Bisulfite changes unmodified C to U (read as T), while m5C is shielded and reads as C, giving base-resolution m5C quantification.
Arraystar Single-Nucleotide tRNA m5C Modification Seq(BS-seq) is an end-to-end sample-to-data service for tRNA, from RNA sample QC and tRNA treatment through library construction, sequencing, and bioinformatics. Sites are localized within mature tRNA transcripts with relative coordinates, tRNA gene attribution, anticodon and isotype, and structural-domain localization.
Benefits
In tRNA, m5C contributes to structure and function, so site-level mapping is valuable. Bisulfite converts unmodified C to U (read as T) while m5C is shielded and read as C, resolving each m5C site at base resolution.
Single-base resolution: pinpoints m5C sites in tRNA.
Quantitative measurement: the conversion read-out yields reliable modification fractions.
No antibody dependence: eliminates enrichment bias and cross-reactivity concerns.
Functional annotation: Sites are localized within mature tRNA transcripts with relative coordinates, tRNA gene attribution, anticodon and isotype, and structural-domain localization.
Application-ready: supports decoding fidelity, codon use, aminoacylation, and tRNA structural biology across tRNA isotypes.
| Service Name | Price |
|---|---|
| Single-Nucleotide tRNA m5C Modification Seq(BS-seq) |
Background
In tRNA, the modification stabilizes the folded L-shaped structure and fine-tunes anticodon–codon decoding, so base-resolution mapping within maturation loops and the anticodon is what distinguishes it. Bisulfite conversion deaminates unmodified cytidine to uracil (read as thymine) while 5-methylcytidine is protected and read as cytosine, mapping each m5C site at single-base resolution. This antibody-free readout maps each m5C site at single-base resolution.
The method was validated for tRNA, sites are localized within mature tRNA transcripts with relative coordinates, tRNA gene attribution, anticodon and isotype, and structural-domain localization. (Dai Q, et al. He C. Nature Biotechnology 2024;42(10):1559-1570., DOI: 10.1038/s41587-023-02034-w).
Key concept: Single-Nucleotide tRNA m5C Modification Seq(BS-seq) is a base-resolution method in which bisulfite conversion turns unmodified C into U (read as T) while m5C is protected and read as C, mapping and quantifying m5C at single-base resolution.
Figure 1. BS-seq m5C detection scheme. Bisulfite converts unmodified C to U (interpreted as T), while m5C is shielded and reads as C, giving base-resolution m5C quantification.
Workflow
A streamlined workflow carries the sample to base-resolution 5-methylcytidine (m⁵C) datasets:
Figure 2. Single-Nucleotide tRNA m5C Modification Seq workflow.
Bioinformatics
Downstream analysis maps reads to a curated reference, calls C-to-T conversion signatures, and reports m5C stoichiometry per position with motif, distribution, differential, enrichment, and genome-browser results.
Deliverables
- Raw m⁵C tRNA sequencing FASTQ data files, formatted for GEO/SRA public database submission.
- QC report with BAM alignment files and key mapping metrics.
- High-confidence single-base m⁵C tRNA site tables (XLSX) with coordinates, anticodon, isotype, and stoichiometry.
- Single-base m⁵C distribution plots across tRNA domains and consensus m⁵C motif logos (PDF/PNG).
- Single-base resolution differential m⁵C tRNA site tables (XLSX) with fold change and significance.
- Genome browser-compatible m⁵C signal track files (bigWig/bedGraph) and a structured tRNA report.
Research Applications
- tRNA decoding fidelity: maps the modification within anticodon and wobble regions.
- Codon use & aminoacylation: relates base-resolution modification to charging and translation.
- tRNA structural biology: localizes sites within D-loop, anticodon, TΨC, and acceptor stem.
- Isotype-resolved analysis: compares modification across tRNA isotypes and isoacceptors.
- Disease-oriented studies: finds differentially modified tRNA sites as biomarkers.
References
[1] Frommer M, McDonald LE, Millar DS, et al. Paul CL. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. PNAS 1992;89(5):1827-1831. DOI: 10.1073/pnas.89.5.1827.
Sample Requirements
Sample Storage
For cells/tissue: use TRIzol or an RNA-stabilizing reagent, quick-freeze in liquid nitrogen, and keep at –80 °C.
For RNA: dissolve in ethanol or RNase-free water, store at –80 °C, and limit freeze–thaw cycles.
Dispatch Guidelines
Transfer each sample into a 1.5 mL nuclease-free tube.
Close the tube securely with parafilm or a cap lock to preserve integrity.
Send on dry ice with sufficient insulation to sustain the required temperature.
| Sample Type | Notes |
|---|---|
| Whole blood | Use EDTA tubes only, as heparin is not compatible with subsequent analytical procedures. |
| Cultured cells | Submission of cell pellets is preferred to ensure high-quality material for processing. |
| Tissue | Provide fresh or frozen specimens, ensuring that necrotic material is strictly avoided. |
| Total RNA | Maintain an OD 260/280 ratio ≥ 1.8 and RIN ≥ 7 with no visible degradation. Submit at least 10 µg total RNA. |
FAQ
Which tRNA features are annotated?
Sites are localized within mature tRNA transcripts with relative coordinates, tRNA gene attribution, anticodon and isotype, and structural-domain localization. This lets you interpret each site in the context of its host tRNA isotype and structural domain. The method supports both mRNA and tRNA workflows, so you can choose the readout that matches your study.
Does this method measure the modification at single-base resolution?
Yes. Bisulfite conversion deaminates unmodified cytidine to uracil (read as thymine) while 5-methylcytidine is protected and read as cytosine, mapping each m5C site at single-base resolution. This provides single-base resolution and absolute stoichiometry for each site in tRNA.
How does this method differ from antibody-based tRNA profiling?
Antibody-based methods rely on enrichment and offer limited resolution with intrinsic motif bias. This method is antibody-free and delivers base-resolution, quantitative calling in tRNA, avoiding cross-reactivity and enrichment bias. This suits projects needing quantitative site-level mapping rather than region-level enrichment, and it pairs well with orthogonal validation.
How does this service support research on m5C modification?
By profileping m5C at base resolution, the service helps researchers examine its regulatory roles in RNA stability and expression, supporting high-impact studies.
Why is single-base m5C profiling valuable?
Precise m5C detection provides reliable position-level evidence that underpins strong conclusions and advances the field.
!This assay is provided for research applications only and is not intended for clinical diagnosis, treatment, or personal health evaluation.